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Plant Physiology, August 1999, Vol. 120, pp. 1043–1048, www.plantphysiol.org © 1999 American Society of Plant Physiologists
The Endogenous Sulfated Pentapeptide Phytosulfokine-a
Stimulates Tracheary Element Differentiation of
Isolated Mesophyll Cells of Zinnia1
Yoshikatsu Matsubayashi*, Leiko Takagi, Naomi Omura, Akiko Morita, and Youji Sakagami
Laboratory of Bioactive Natural Products Chemistry, Graduate School of Bioagricultural Sciences, Nagoya
University, Chikusa, Nagoya 464–8601, Japan
Dispersed zinnia (Zinnia elegans) mesophyll cells cannot differentiate into tracheary elements (TEs) at low cell density conditions
even if auxin and cytokinin are present in the medium, indicating
the involvement of intercellular interactions during the initiation
and/or subsequent progresses in TE differentiation. When zinnia
cells were incubated at a low density (2.5 3 104 cells mL21) in
TE-inductive medium in the presence of various concentrations of
phytosulfokine (PSK)-a, which was originally identified as an intercellular signal peptide involved in cell proliferation, TE differentiation was strongly stimulated in a dose-dependent fashion; more than
35% of the living cells differentiated into TEs by 5 d of culture in the
presence of 10 nM PSK-a. Enzyme-linked immunosorbent assay and
mass spectroscopy confirmed that cultured zinnia cells produce
nanomolar levels of PSKs under inductive conditions. These results
suggest that PSK-a is a factor responsible for TE differentiation of
zinnia mesophyll cells.
TE formation in culture has been used as a model system
for the study of cell differentiation of higher plants (for
review, see Fukuda, 1992). In mechanically isolated mesophyll cells of zinnia (Zinnia elegans) in liquid culture,
differentiation into TEs can be readily induced by phytohormones and clearly distinguished on the basis of morphological features (Fukuda and Komamine, 1980a). Because of the nature of this system, individual cells respond
homogeneously to physiological and chemical stimuli, and
synchronously differentiate at relatively high frequency.
Many researchers have investigated the determining factor(s) of TE differentiation at the cellular level and found
that differentiation necessitates at least two exogenous factors, an auxin and a cytokinin (for review, see Fukuda and
Komamine, 1985). In particular, cytokinins have been considered an absolute requirement in zinnia mesophyll cells
(Fukuda and Komamine, 1980a).
The initial cell density is also a determining factor in TE
differentiation in zinnia mesophyll cell cultures (Fukuda
and Komamine, 1980a). Differentiation occurs synchronously at high frequency above an initial cell density of
4.2 3 104 cells mL21, but is significantly suppressed below
1
This research was supported by the Program for Promotion of
Basic Research Activities for Innovative Biosciences.
* Corresponding author; e-mail [email protected]; fax
81–52–789 – 4118.
this threshold, suggesting that intercellular interactions are
involved in the initiation and/or subsequent progresses in
TE differentiation. An oligosaccharide-like factor in zinnia
conditioned medium appear to play an important role in
cell expansion and metaxylem-like TE differentiation (Roberts et al., 1997), but the active principle has not been
chemically identified.
The relative growth rate of the plant cells in culture also
strictly depends on the initial cell density, even if sufficient
amounts of auxins and cytokinins are present in the medium, so additional factors must play a role in cell proliferation (Bellincampi and Morpurgo, 1987; Birnberg et al.,
1988). In 1996, we first isolated one of these factors from
conditioned medium derived from mesophyll culture of
asparagus, and determined its structure to be a sulfated
pentapeptide, H-Tyr(SO 3 H)-Ile-Tyr(SO 3 H)-Thr-Gln-OH
(Matsubayashi and Sakagami, 1996). This peptide, named
PSK-a, compensates for cell growth suppression in lowdensity cultures at a concentration as low as 1.0 nm. PSK-a
has also been identified in conditioned medium derived
from rice and maize cell cultures, apparently promoting
cell growth by interacting with specific binding sites distributed upon plasma membranes (Matsubayashi et al.,
1997; Matsubayashi and Sakagami, 1999).
Although the generality of PSK-a in plant kingdom has
not yet been well clarified, its influence on cell differentiation and proliferation are clearly of interest. In the present
study, we investigated the physiological effects of PSK-a
on TE differentiation and proliferation of zinnia mesophyll
cells, and also determined, using ELISA and MS, whether
zinnia cells themselves produce PSK-a.
MATERIALS AND METHODS
Materials
Sep-Pak cartridges were purchased from Millipore and
Develosil ODS-5 and Develosil ODS-10 reverse-phase columns were purchased from Nomura Chemicals (Seto, Japan). Twenty four-well microplates were obtained from
Iwaki (Chiba, Japan). Polystyrene, 96-well plates for ELISA
were from Nunc (Naperville, IL). All of the other inorganic
and organic chemicals were obtained from Wako Pure
Abbreviations: PSK, phytosulfokine; TE, tracheary element;
TFA, trifluoroacetic acid.
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1044
Matsubayashi et al.
Chemicals (Osaka). PSK-a was prepared by solid-phase
synthesis as previously described (Matsubayashi et al.,
1996).
Plants
Seeds of zinnia (Zinnia elegans cv Canary bird; Takii
Shubyo, Kyoto) were grown on moist sterile soil at 25 6
2°C with a 16-h light period (approximately 20,000 lux at
the plant level).
Isolation of Single Cells from the Mesophyll
Zinnia leaves were sterilized for 10 min in a solution of
0.05% (w/v) NaOCl containing 0.05% (w/v) Tween 20,
then rinsed three times with sterile water. Single cells were
liberated by homogenization with a glass homogenizer in
0.2 m mannitol solution. The homogenate was filtered
through a 37-mm stainless-steel mesh, and the filtrate was
centrifuged at 100g for 3 min. The separated single cells
were washed with 0.2 m mannitol three times and used in
the following experiments.
Cell Culture and Bioassay Methods
Plant Physiol. Vol. 120, 1999
ously (Matsubayashi et al., 1999). Two different PSK-a conjugated proteins, Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-(Gly)3Cys-linker-KLH (antigen A) and Tyr(SO3H)-Ile-Tyr(SO3H)Thr-Gln-(Ala)3-Lys-linker-BSA (antigen B) were prepared by
coupling the peptides with the corresponding proteins. Rabbits were immunized with antigen A, and the obtained
antibodies were purified with an immunoaffinity column
containing Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-Cys-linkerresin. For quantification of PSK-a, polystyrene 96-well plates
were coated with antigen B and blocked with 0.1% (w/v)
BSA in PBS (10 mm phosphate buffer [pH 7.0] containing
8.0 g L21 NaCl). Purified antibody and samples were then
added and the plates were incubated at 37°C for 1.5 h.
During this period, competition occurred for the antibody
between antigen B bound to the plate and free PSK-a in
solution. After washing with PBS containing 0.1% (w/v)
Tween 20, plates were incubated for 1.5 h with a solution of
horseradish peroxidase-coupled anti-rabbit immunoglobulin. After three washings, orthophenylenediamine solution
containing 0.01% hydrogen peroxide was added, and the
plates were incubated for 20 min at 37°C. Color development was terminated with sulfuric acid, and optical density was recorded at a wavelength of 490 nm with a plate
reader.
The basal medium used for TE differentiation experiments was prepared according to the method of Fukuda
and Komamine (1980a), except that NH4Cl was eliminated
to improve the cell viability under low-density conditions.
The medium was adjusted to pH 5.5 with 1.0 n KOH.
Isolated single mesophyll cells were suspended in 0.2 m
mannitol, adjusted to twice the final cell density using a
hemocytometer, and dispensed into 24-well microplates at
a volume of 250 mL per well. Culture medium (125 mL)
prepared at a 4-fold concentration and various sample
solutions (125 mL) were sterilized by filtration and then
added to the cell suspension in each well. The plates were
sealed with laboratory film to avoid evaporation of the
medium, and were then incubated in the dark at 25°C with
continuous rotary shaking at 120 rpm. The TE differentiation frequency was determined by counting the numbers of
undifferentiated and differentiated cells under an inverted
microscope. The TE differentiation frequency for each well
was calculated by dividing the number of TE-differentiated
cells by the total number of undifferentiated and differentiated cells. The cell viability for each well was calculated
by dividing the number of living cells (determined by
bromphenol blue; Suzuki et al., 1992) by the number of
total cells.
For the preparation of conditioned medium, single zinnia mesophyll cells were suspended in 200 mL of culture
medium at a density of 2.0 3 105 cells mL21. This suspension was cultured in 500-mL Erlenmeyer flasks in the dark
at 25°C with rotary shaking at 120 rpm. After 6 d of culture,
conditioned medium was collected by filtration and stored
at 220°C until use.
Conditioned medium derived from zinnia suspension
cultures (400 mL) was concentrated to one-third volume,
adjusted to pH 8.0 with 6.0 n KOH, and then applied to a
DEAE Sephadex A-25 column (3.2 3 12 cm, flow rate 100
mL h21) equilibrated with 20 mm Tris-HCl buffer, pH 8.0.
The column was washed with 200 mL of the buffer, and
fractions were eluted successively with 200 mL of this
buffer containing 400, 800, or 1,200 mm KCl. Fractions of
800 and 1,200 mm were concentrated to one-half volume
and TFA was added to this combined fraction at a final
concentration of 0.1% (v/v). This solution was applied to
C18 cartridges (Sep-Pak Vac, 12 mL 3 2, flow rate 150 mL
h21) that had been equilibrated with 0.1% TFA. After washing with 80 mL of 0.1% (v/v) TFA in water, fractions were
eluted with 30% (v/v) acetonitrile containing 0.1% (v/v)
TFA. After lyophilization, materials were dissolved in 1.0
mL of 20 mm KH2PO4-KOH buffer, pH 5.8, and then applied to a Bio-Gel P-2 extra-fine column (1.7 3 42 cm),
previously equilibrated with the same buffer. Fivemilliliter fractions were collected and assayed by ELISA.
Positive fractions recovered from the Bio-Gel column were
lyophilized, dissolved in 200 mL of 10% (v/v) acetonitrile in
0.1% TFA, and chromatographed on a Develosil ODS-5
column (4.6 3 250 mm, Nomura Chemicals) by isocratic
elution with 10% (v/v) acetonitrile in 0.1% TFA at a flow
rate of 1.0 mL/min with monitoring of the UV A220. Fractions were collected every 1.0 min and assayed by ELISA
after lyophilization.
Competition ELISA Procedure
MS
Preparation of anti-PSK-a polyclonal antibodies and the
procedure for competitive ELISA were described previ-
Positive fractions determined by ELISA were dissolved
in 20 mL of water and directly analyzed with a vacuum
Purification of PSK-a from Conditioned Medium
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Tracheary Element Differentiation of Zinnia Cells Stimulated by a Sulfated Peptide
1045
generator platform quadrupole mass spectrometer (Fisons,
Cheshire, UK) equipped for electrospray ionization. The
source temperature was maintained at 70°C, and the range
m/z 50 to 1,000 was scanned over 1.9 s.
RESULTS AND DISCUSSION
Effects of Initial Cell Density on TE Differentiation
Although Fukuda and Komamine’s medium (Fukuda
and Komamine, 1980a) has been widely used in TE differentiation experiments, zinnia cells cultured at low cell density often show relatively low viability in this medium
(approximately 35% at 2.5 3 104 cells mL21). This phenomenon is likely attributed to ammonium ions, because
cell growth of mesophyll primary cultures under low cell
density conditions is strongly inhibited by ammonium ions
even if sufficient amounts of PSK-a are added to the
medium (Matsubayashi and Sakagami, 1998). Therefore,
we modified Fukuda and Komamine’s medium to eliminate NH4Cl.
The TE differentiation frequency of single zinnia cells in
suspension culture was markedly dependent on the initial
cell density (Fig. 1). The TEs formed at relatively high
frequency at the initial cell density of 1.0 3 105 and 5.0 3
104 cells mL21, reaching approximately 30% after 5 d of
culture. In contrast, TE differentiation was markedly inhibited at densities below 2.5 3 104 cells mL21, and the frequencies remained approximately 5% or less of these values even after 5 d of culture. A similar observation was
made in a previous report (Fukuda and Komamine, 1980a),
suggesting the presence of intercellular communication
mediated by chemical, if not physical, signals.
Effects of PSK-a on TE Differentiation
Figure 2. Effects of PSK-a on TE differentiation (A) and cell viability
(B). Single zinnia cells were incubated in liquid medium at a density
of 2.5 3 104 cells mL21 in the presence of various concentrations of
PSK-a. The TE differentiation frequency for each well was calculated
by dividing the number of TEs by the total number of living cells, and
cell viability was calculated by dividing the number of living cells by
the number of total cells. Data are means of three replicates 6 SD. M,
1.0 mM; ●, 0.1 mM; ‚, 0.01 mM; f, 1.0 nM; E, 0.1 nM; Œ, control.
On the assumption that a chemical factor(s) other than
auxin or cytokinin is involved in TE differentiation, we
Figure 1. Effects of the initial cell density on TE differentiation. Single
zinnia cells were suspended in liquid medium, dispensed into 24well culture plates at a final volume of 500 mL per well, and incubated at 25°C with shaking at 120 rpm. The TE differentiation frequency for each well was calculated by dividing the number of TEs
by the total number of living cells. Data are mean values of three
replicates 6 SD. M, 1 3 105 cells/mL; ●, 5 3 104 cells/mL; ‚, 2.5 3
104 cells/mL; f, 1.3 3 104 cells/mL; E, 6.2 3 103 cells/mL; Œ, 3.1 3
103 cells/mL.
investigated the effects of PSK-a, an endogenous peptide
growth factor shown to compensate for growth suppression observed at low cell density, on TE differentiation of
zinnia cells.
When the cells, cultured at a density of 2.5 3 104 cells
mL21, were treated with PSK-a at a concentration range
from 0.1 nm to 1.0 mm, TE differentiation was strongly
stimulated in a dose-dependent manner (Fig. 2A). The
TE-formation-stimulating activity of PSK-a was detected at
concentrations as low as 0.01 mm, where more than 35% of
the living cells differentiated into TEs by 5 d of culture in
the presence of PSK-a. In contrast, the frequency of TE
differentiation in the absence of PSK-a remained at approximately 1% by 5 d, indicating that PSK-a compensates for
the suppression of TE differentiation observed at low cell
densities.
Zinnia mesophyll cells have an absolute requirement for
an auxin and a cytokinin for TE differentiation (Fukuda
and Komamine, 1980a). To determine how these two phytohormones and PSK-a are involved in the stimulation of
TE differentiation, we cultured mesophyll cells in media
that contained 1.0 mm PSK-a and four combinations of
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1046
Matsubayashi et al.
plant hormones: NAA and 6-BA, NAA only, 6-BA only,
and no hormones. Elimination of NAA and/or BA completely suppressed TE differentiation (0%) even when the
culture medium contained a sufficient amount of PSK-a for
the stimulation. We conclude that PSK-a requires both
auxin and cytokinin to stimulate TE differentiation of zinnia cells.
TE differentiation stimulated by PSK-a is not a secondary effect caused by inhibiting cell death, because cell
viabilities were not altered by changing the PSK-a concentration (Fig. 2B). It is also unlikely that PSK-a promoted the
differentiation by increasing a cell division, because more
than 80% of TEs differentiated directly from the dispersed
mesophyll cells without intervening cell division (Table I;
Fig. 3). Direct TE differentiation without intervening cell
division has been reported in many studies of colchicinetreated (Fukuda and Komamine, 1980b) or g-irradiated
cells (Phillips, 1981; Sugiyama et al., 1986), as well as after
serial observation of single mesophyll cells (Fukuda and
Komamine, 1980b).
The fact that the minimum density required for zinnia
cell division in this medium was approximately 103 cells
mL21, which is far lower than that required for TE differentiation, further supports the independence of differentiation and proliferation. At a density of 2.5 3 104 cells
mL21, more than 90% of the viable cells that had not
differentiated into TEs had divided by 5 d of culture regardless of the concentration of PSK-a. Thus, it may be
concluded that PSK-a stimulated TE differentiation by recruiting more cells into the TE developmental pathway
without altering cell viability or cell division.
Identification of PSKs in Conditioned Medium
Derived from Zinnia Suspension Culture
Because PSK-a stimulates TE differentiation of zinnia
mesophyll cells, we next investigated whether zinnia mesophyll cells themselves produce PSK-a. As a first step, we
tested the effects of crude conditioned medium on TE
differentiation by adding aliquots to bioassay media. As
shown in Figure 4, TE differentiation was stimulated (compared with control) by conditioned medium; the maximum
Plant Physiol. Vol. 120, 1999
Figure 3. Micrographs of zinnia mesophyll cells cultured in the
presence or absence of PSK-a. Single zinnia cells were incubated for
5 d in medium containing PSK-a at a concentration of 10 nM (A) or
in the absence of PSK-a (B). Bar 5100 mm.
frequency was about 10% after 8 d of the start of bioassay
when the conditioned medium concentration was 12.5%
(v/v), indicating that zinnia conditioned medium may contain PSK-a or PSK-a-like compounds. In contrast, the addition of conditioned medium at a concentration above
12.5% resulted in inhibition of TE differentiation (data not
shown). Conditioned medium may contain inhibitory factor(s) for TE differentiation as well as stimulatory factor(s).
Table I. Percentages of TEs differentiated without intervening cell
division
Zinnia mesophyll cells were cultured at an initial density of 2.5 3
104 cells mL21 in media containing PSK-a at various concentrations.
The TE differentiation frequency was determined after 5 d of culture.
Data are mean values of three replicates 6 SD.
PSK-a
Concentration
1.0 mM
0.1 mM
0.01 mM
1.0 nM
0.1 nM
None
a
ND, Not determined.
TEs
% of living cells
% of total TEs
39 6 7
39 6 2
35 6 2
16 6 5
864
162
81 6 7
87 6 2
82 6 4
86 6 3
NDa
ND
Figure 4. Effects of conditioned medium on TE differentiation. Single
zinnia cells were incubated in liquid medium at a density of 2.5 3
104 cells mL21 in the presence of various concentrations of conditioned medium. Data are the means of three replicates 6 SD. M,
12.5%; ●, 3.2%; ‚, 0.8%; f, control.
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Tracheary Element Differentiation of Zinnia Cells Stimulated by a Sulfated Peptide
Figure 5. Identification of PSKs in conditioned medium derived from
zinnia mesophyll cell culture. A, HPLC profile of purified conditioned medium. Conditioned medium derived from TE-inductive
culture was separated by two steps of open-column chromatography
and reverse-phase HPLC. Fractions were collected every minute. B,
Results of competitive ELISA. The amount of PSKs contained in each
fraction was determined by competitive ELISA based on anti-PSK-a
antibodies. Peaks eluting at 10.2 and 15.7 min were estimated to be
PSK-a and PSK-b, respectively. C, Comparison of mass spectrum of
natural sample and synthetic PSK-a. A combined fraction (9.0–11.0
min) was concentrated and analyzed by MS (1st row). A pseudomolecular ion of m/z 845 corresponds to [M-H]2 and a fragment ion of
m/z 765 corresponds to [M-H-80]2 of PSK-a, coinciding well with
the spectrum of synthetic PSK-a (2nd row). A fraction (15.0–16.0
min) was concentrated and analyzed by MS (3rd row). A pseudomolecular ion of m/z 717 corresponds to [M-H]2, and a fragment ion of
m/z 637 corresponds to [M-H-80]2 of PSK-b, coinciding well with
the spectrum of synthetic PSK-b (4th row).
To determine if conditioned medium contains PSK-a, we
fractionated zinnia conditioned medium by stepwise elution from a DEAE Sephadex column. Fractions of 800 and
1,200 mm KCl were desalted on a Sep-Pak column and on
Bio-Gel P-2 by gel-permeation chromatography. Each
eluted fraction (5.0 mL) was assayed by ELISA, and positive fractions (40–55 mL) were pooled and lyophilized.
These fractions were further purified by reverse-phase
HPLC, with monitoring of UV A220 (Fig. 5A). Fractions (1.0
mL) were collected from 0 to 20 min and assayed by ELISA.
Significant amounts of PSKs were detected in the 9.0 to 11.0
min and the 15.0 to 16.0 min fractions (Fig. 5B). A combined
fraction of 9.0 to 11.0 min had a pseudomolecular ion of
m/z 845 corresponding to [M-H]2 and a fragment ion of
m/z 765 corresponding to [M-H-80]2, as shown by MS,
confirming that this fraction contains PSK-a (Fig. 5C). By
1047
comparing the retention time of eluted peak and that of
synthetic PSK-a (data not shown), a peak eluting at 10.2
min was determined to be PSK-a. A similar procedure
showed that a peak eluting at 15.7 min was PSK-b, a
C-terminal truncated peptide of PSK-a. A control experiment revealed that the total recovery of PSK-a by this
purification procedure was 15%. Therefore, the total
amount of PSKs contained in 400 mL of conditioned medium was estimated to be 3.0 nmol equivalent to PSK-a.
We have shown that PSK-a stimulates TE differentiation
of dispersed zinnia mesophyll cells without intervening
cell division in the presence of auxin and cytokinin. We
also confirmed that zinnia cells in culture produce considerable amounts of PSK-a. Although this peptide was originally isolated as a mitogenic factor from conditioned medium derived from an asparagus mesophyll culture
(Matsubayashi and Sakagami, 1996), current results indicate that PSK-a stimulates cell differentiation instead of cell
division under the specified conditions.
What is the fundamental function of PSK-a? One possibility is that PSK-a makes cells receptive to signals that
ultimately determine the cell destiny, i.e. cell division or
cell differentiation. Research into PSK-a receptors transmitting secondary messages that activate a specific set of
genes appears to be warranted.
ACKNOWLEDGMENTS
We thank Dr. Hiroo Fukuda and Hiroyasu Motose (Graduate
School of Sciences, University of Tokyo) for useful discussions.
Received February 11, 1999; accepted April 30, 1999.
LITERATURE CITED
Bellincampi D, Morpurgo G (1987) Conditioning factor affecting
growth in plant cells in culture. Plant Sci 51: 83–91
Birnberg PR, Somers DA, Brenner ML (1988) Characterization of
conditioning factors that increase colony formation from black
Mexican sweet corn protoplasts. J Plant Physiol 132: 316–321
Fukuda H (1992) Tracheary element formation as a model system
of cell differentiation. Int Rev Cytol 136: 289–332
Fukuda H, Komamine A (1980a) Establishment of an experimental
system for the study of tracheary element differentiation from
single cells isolated from the mesophyll cells of Zinnia elegans.
Plant Physiol 65: 57–60
Fukuda H, Komamine A (1980b) Direct evidence for cytodifferentiation to tracheary elements without intervening mitosis in a
culture of single cells isolated from the mesophyll of Zinnia
elegans. Plant Physiol 65: 61–64
Fukuda H, Komamine A (1985) Cytodifferentiation. In IK Vasil,
ed, Cell Culture and Somatic Cell Genetics of Plants, Vol 2.
Academic Press, New York, pp 149–212
Matsubayashi Y, Hanai H, Hara O, Sakagami Y (1996) Active
fragments and analogs of the plant growth factor, phytosulfokine: structure-activity relationships. Biochem Biophys Res
Commun 225: 209–214
Matsubayashi Y, Morita A, Matsunaga E, Furuya A, Hanai N,
Sakagami Y (1999) Physiological relationships between auxin,
cytokinin, and a peptide growth factor, phytosulfokine-a,
in stimulation of asparagus cell proliferation. Planta 207:
559–565
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1999 American Society of Plant Biologists. All rights reserved.
1048
Matsubayashi et al.
Matsubayashi Y, Sakagami Y (1996) Phytosulfokine, sulfated
peptides that induce the proliferation of single mesophyll
cells of Asparagus officinalis L. Proc Natl Acad Sci USA 93:
7623–7627
Matsubayashi Y, Sakagami Y (1998) Effects of the medium
ammonium-nitrate ratio on competence for asparagus cell division induced by phytosulfokine-a. Plant Cell Rep 17: 368–372
Matsubayashi Y, Sakagami Y (1999) Characterization of specific
binding sites for a mitogenic sulfated peptide, phytosulfokine-a,
in the plasma membrane fraction derived from Oryza sativa L.
Eur J Biochem (in press)
Matsubayashi Y, Takagi L, Sakagami Y (1997) Phytosulfokine-a,
a sulfated pentapeptide, stimulates the proliferation of rice cells
by means of specific high- and low-affinity binding sites. Proc
Natl Acad Sci USA 94: 13357–13362
Plant Physiol. Vol. 120, 1999
Phillips R (1981) Direct differentiation of tracheary elements in
cultured explants of gamma-irradiated tubers of Helianthus tuberosus. Planta 153: 262–266
Roberts AW, Donovan SG, Haigler CH (1997) A secreted factor
induces cell expansion and formation of metaxylem-like tracheary elements in xylogenic suspension cultures of zinnia. Plant
Physiol 115: 683–692
Sugiyama M, Fukuda H, Komamine A (1986) Effects of nutrient
limitation and g-irradiation on tracheary element differentiation
and cell division in single mesophyll cells of Zinnia elegans. Plant
Cell Physiol 27: 601–606
Suzuki K, Ingold E, Sugiyama M, Fukuda H, Komamine A (1992)
Effects of 2,6-dichlorobenzonitrile on differentiation to tracheary
elements of isolated mesophyll cells of Zinnia elegans and formation of secondary cell walls. Physiol Plant 86: 43–48
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